Deoxyribonucleoside Kinases
In eukaryotic cells there exist at least three deoxyribonucleoside kinases, thymidine kinase 1 and 2, TK and TK2, deoxycytidine kinase, dCK and deoxyguanosine kinase, dGK.
There exist two human gene loci encoding the two different thymidine kinases. Thymidine kinase can be found in the cytosol of the cell. Thymidine kinase 2 is localized in the mitochondrion and participates in mitochondrial DNA synthesis. TK2 has a different amino acid sequence, substrate specificity and expression profile from thymidine kinase 1.
Thymidine kinase 1 activity is most pronounced from the end of the G1 phase and through the S phase of the cell cycle. TK and dCK are responsible for regulating the pools of TTP and dCTP in the cell through the salvage pathway. The TK enzyme is the enzyme that catalyses the transformation of thymidine to thymidine monophosphate (TMP) in the presence of adenosine triphosphate (ATP).
Deoxycytidine kinase, activity is found during the whole cell cycle and is primarily identified in cells of lymphoid origin. TK2 is located in the mitochondrion and has an almost equal affinity for dC as dCK.
Diagnosis of Disease
In the early 1980-ies it was shown that thymidine kinase activity not only can be detected in serum from patients affected with various tumour diseases but varied extensively from healthy human individuals that normally exhibit very low TK activity
The use of TK as a single tumour marker for the diagnosis and prognosis of haematological cancer diseases is now widely accepted. In certain instances TK has shown direct diagnostic capabilities.
The activity of TK and other deoxyribonucleoside kinases was early correlated to disease, which is summarised in reviews by Eriksson et al. 2002 and by Topolcan and Holubec 2008.
TK2 has been connected to mitochondrial diseases.
Assays for Determination of Deoxyribonucleoside Kinases
Radioactive substrates, such as 3H-thymidine, have been widely used for determination of TK and dCK activity in the filter-binding assays. For routine clinical diagnostics, radioactive based assays are not an attractive option.
During the last years several attempt has been made to develop non-radioactive based assays, all of these better in one way or the other. Below follow a set of methods, assays and reagent compositions used in the detection of different deoxyribonucleoside kinases.
In EP1385005 a non-radioactive TK activity based assay based on competitive mono-phosphorylation of azidothymidine (AZT) is presented. This technology has been further developed into an automated 2-step LIAISON® luminometric diagnostic assay by Diasorin S.r.I. In this two-step assay, AZT is first phosphorylated to monophosphorylated AZTMP by the TK present in a sample. Then luminescence is next detected from AZTMP conjugated to isoluminol (AZTMP-ABEI) which is competing with the TK phosphorylated AZTMP on solid surface substrate bound anti-AZTMP antibodies. The major drawback of this two-step assay is the limited dynamic reading. Another drawback is the use if modified nucleoside substrate in place of the natural thymidine.
In WO2009/063254 a semi-homogenous method for the determination of TK activity in a sample by the use of monophosphorylated Br-dU which is separated by HPLC and detected by UV is disclosed. Disadvantage with this method is that the reaction is done with a modified substrate and the detection is done in a separate step.
US2008/248472 discloses a solid phase assay utilizing complementation of TK activity, in a sample, by deoxyribonucleoside kinases in the reagent mix for production of Br-dUTP, which is a derivative of the natural TTP. The Br-dUTP is subsequently incorporated by primer extension using a RNA-dependent DNA polymerase. The solid phase bound RNA template is build of the monomer rA and is 200-400 monomers long, and attached to solid phase. Incorporated [Br]U is detected in the second step using an anti-[Br]dU alkaline phosphatase conjugated antibody in an ELISA for final determination of TK activity. This two-step assay is cumbersome to perform with many manual interaction steps and takes excessively long time to execute.
Reliable assays for measuring dCK activity in a sample have not yet been presented.